DNA aptamers specific to CD2000R1 and their therapeutic uses

ABSTRACT

Disclosed are short DNA aptamers that selectively recognize CD200R1, a protein expressed on the surface of myeloid and lymphoid cells that delivers immune inhibitory signals to modulate inflammation when engaged with its ligand, CD200. Also disclosed is the use of said aptamers as therapeutic agents, for the purpose of decreasing inflammatory response; treatment of spinal cord injury; treatment of an immune related disease such as arthritis, asthma, allergy, infection; as a course of treatment during or after transplantation; or for treatment of an autoimmune disorder such as systemic lupus erythematosus, Parkinson&#39;s Disease, or multiple sclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/127,909, filed on Sep. 21, 2016, which is a 35 U.S.C. § 371national stage filing of International Application No.PCT/CA2015/050212, filed on Mar. 20, 2015, which claims the benefit ofpriority of U.S. Provisional Application No. 61/968,740, filed on Mar.21, 2014. The contents of each of the foregoing applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

This invention relates to the discovery and uses of a class of short DNAaptamers that selectively recognize CD200R1, a protein expressed on thesurface of myeloid and lymphoid cells that delivers immune inhibitorysignals to modulate inflammation when engaged with its ligand, CD200.

CD200R1

CD200R1 is a type I glycoprotein expressed on cells of myeloid andlymphoid lineage. It delivers immune inhibitory signals upon ligation tothe widely distributed cell surface glycoprotein CD200. Structurally,CD200R1 contains two Ig-like domains, a transmembrane region, and acytoplasmic tail containing a NXPY motif which is phosphorylated uponCD200 ligation inducing recruitment of the adaptor protein Dok2 andsubsequent signal transduction.

The physiological importance of CD200:CD200R1 inhibitory signalling hasbeen established in a number of diseases including arthritis,transplantation, and a number of central nervous system autoimmunediseases such as Parkinson's disease (PD) and multiple sclerosis (MS).For instance, a recombinant CD200.Fc fusion protein has been shown tobehave as a potent in-vivo immunosuppressant, prolonging allo- andxenograph survival as well as suppressing collagen-induced arthritis inmice. Also, the inhibition of CD200:CD200R1 signalling on microglialcells using a blocking antibody to CD200R1 exacerbated neurodegenerationand disease state in a murine model of experimental autoimmuneencephalomyelitis (EAE). These findings were further supported in aseparate EAE study where treatment with CD200.Fc suppressed microglialaccumulation, and decreased the production of pro-inflammatory cytokinesIL-6, TNF-α, and nitric oxide by myeloid cells in the spleen and centralnervous system.

CD200R1 signalling has also been implicated in tissue specificautoimmunity, as both systemic and local treatment with an anti-CD200R1agonistic antibody suppressed experimental autoimmune uveitis (EAU), amodel of CD4⁺ T-cell organ-specific autoimmunity of the eye.

Thus the development of safe and effective immunomodulatory agents whichstimulate CD200R1 signalling are of clinical interest. Despite advancesin antibody and protein engineering, the major drawbacks ofprotein-based CD200R1 stimulators are their immunogenicity arising fromtheir chronic use and their production costs resulting in expensivetherapies for patients.

It would be useful to have a non-protein composition that binds toCD200R1 with high specificity and/or affinity without immune responses.Such a composition may act as a stimulator of immune inhibitorysignalling by selectively binding, and hindering the function of, orinactivating, the CD200R1, and thus be useful as a treatment for immunerelated disease such as arthritis, asthma, allergy, infection, as acourse of treatment during or after transplantation, or for treatment ofautoimmune disorders such as systemic lupus erythematosus, Parkinson'sDisease, or multiple sclerosis. Such a composition may also be usefulconjugated or otherwise associated with a cytotoxic agent forspecifically targeting such an agent to a CD200R1 expressing oroverexpressing cell.

Aptamers

Aptamers are short, single-stranded nucleic acid oligomers (ssDNA orRNA) which adopt a specific tertiary structure allowing them to bind tomolecular targets with high specificity and affinities comparable tothat of monoclonal antibodies, through interactions other than classicWatson-Crick base pairing. In some cases, aptamers will displayfunctional properties beyond just binding to their target. For instance,an aptamer to the inflammation factor human neutrophil elastase (hNE)was shown to significantly reduce lung inflammation in rats anddisplayed greater specificity for their target than an antielastase IgGcontrol. Examples of other aptamers exhibiting functional attributesinclude a DNA aptamer to anti-HIV reverse transcriptase and RNA aptamersto the basic fibroblast growth factor and vascular endothelial growthfactor. Finally, a single-stranded DNA aptamer selected to bind tothrombin has been shown to inhibit thrombin-catalyzed fibrin-clotformation in vitro using either purified fibrinogen or human plasma.Thus, aptamers can be derived to either block protein-proteininteractions or act as agonists to cell surface receptors.

Aptamers have been generated for over 100 proteins including growthfactors, transcription factors, enzymes, immunoglobulins, and receptors.A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds itstarget with sub-nanomolar affinity, and discriminates against closelyrelated targets. They have several advantages over antibodies. As aclass, they have demonstrated therapeutically acceptable toxicity, and alack of immunogenicity. Aptamers can typically be administered bysubcutaneous injection due to their low solubility as compared toantibodies. Aptamers are chemically robust, and can be readilymanufactured since they can be chemically synthesized.

In contrast to antibodies and other protein-based agents, aptamers havea number of advantages including a long shelf life, low immunogenicity,and chemical synthesis. However, aptamers as therapeutic entities dodisplay poor pharmacokinetic profiles as unprotected RNA or DNA aptamersare rapidly removed from circulation due to renal filtration andnuclease degradation. However, improved pharmacokinetic properties havebeen observed upon site-specific conjugation of polyethylene glycol(PEG) polymers to aptamer termini as well as the incorporation ofnuclease resistant 2′-F or 2′-Me nucleotides in the case of RNAaptamers. Functional aptamers which target co-stimulatory orco-inhibitory receptors represent a new class of targetedimmunotherapeutic agents with unique and advantageous properties. Thusfar, aptamers with either agonistic or antagonistic function have beendeveloped to a number of immune co-receptors including CTLA-4, 4-1BB,OX-40, IL-6R, IL-10R, and CD28. However, only a few of them have beenvalidated for activity in-vivo.

Membrane impermeant aptamers have the potential to be used asantagonists themselves, or to serve as intracellular delivery agentsspecific to an internalized surface marker on a cancer cell, forexample. Therapeutic cargos such as siRNAs, antisense oligonucleotides,ribozymes as well as low MW drugs, can be directly coupled to aptamersor packaged into particles modified with aptamers. Aptamer-containingconjugates can be constructed by chemically coupling a drug, such as achemotherapeutic drug, to the aptamer via a linker or by intercalatingthe drug into the aptamer folded structure creating a physical complex.The cargo is then imported into a target cell due to the aptamerspecificity while reducing toxicity towards other cells. Cargos can beconjugated to aptamers during solid-phase synthesis or post-synthesis byincorporating an amino or thiol group at one end of the oligonucleotideduring its assembly. A therapeutic protein can also be coupled to theaptamer, to reach an intracellular substrate target. Aptamers can alsobe conjugated to radionuclides or metal chelators to image or kill cellstargeted by the aptamer. Recently, aptamers have been conjugated tonanostructures, representing a promising class of new agents fortargeted imaging and therapy. Thus, cargos can also be encapsulated intosuch nanopaticles decorated on their surface with aptamers. The targetedstructures include nanorods, quantum dots as well as soft and hardnanoparticles.

An aptamer that binds with high specificity and/or affinity to CD200R1would be desirable for its potential as a simple (compared, for example,to an antibody), synthetic, potentially non-immunogenic stimulator ofimmune inhibitory signalling. Such a compound would be useful for avariety of research, diagnostic, and therapeutic uses, for example, forimaging, diagnosis, or for the treatment of immune related disease suchas arthritis, allergy, asthma, infection, as a course of treatmentduring or after transplantation, or for treatment of autoimmunedisorders such as systemic lupus erythematosus, Parkinson's Disease. ormultiple sclerosis. Such a compound could also be useful conjugated orotherwise associated with a cytotoxic agent for specifically targetingsuch an agent to a CD200R1-expressing or overexpressing cell.

Asthma

Asthma is a common long-term inflammatory disease of the airways of thelungs, affecting hundreds of millions of individuals, and causinghundreds of thousands of deaths each year. Symptoms of asthma includeepisodes of coughing, shortness of breath, wheezing, coughing, anddifficulty breathing. Currently, the most common treatment for asthma isinhaled or intranasally administered corticosteroids. They may becombined with long-acting beta agonists, or antileukotriene agents.Effectiveness of these treatments is highly variable. The cause ofasthma is also highly variable, and may include both environmentalfactors and genetic factors. Many of the genes associated or implicatedwith asthma are related to the immune system, or modulatinginflammation.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show that DNA Aptamers selected to bind to theextracellular domain of murine CD200R1 suppress CTL induction in primaryMLC. FIG. 1A: Aptamer sequences which were identified after 15 iterativeSELEX rounds. Full length aptamers consist of an internal variableregion of 25 bp, flanked by two constant regions of 25 bp each. Controlaptamer (cApt) has a scrambled variable sequence with the same constantregions and was used throughout this study. FIG. 1B: DNA aptamers wereadded to 5 day allogeneic-MLC with 2.5×10⁵ C57BL/6 responder cells andan equal number of irradiated BALB/c stimulator cells. CTLs were assayedby ⁵¹Cr release of loaded P815 mastocytoma target cells in 5 hours at a25:1 effector-to-target ratio. Soluble CD200Fc was used as a positivecontrol. Each bar represents average CTL specific lysis±SEM (n=4).Aptamers were added at 22.5 μg/mL (black), 7.5 μg/mL (grey) or 2.5 μg/mL(white). *P<0.01 compared to cApt or no treatment. s\Shown is arepresentative of three independent experiments.

FIGS. 2A, 2B, and 2C show Guided Truncation of CD200R1 aptamers M49 andM52. FIG. 2A: Shown are name, sequence and length of truncated aptamers.Bolded, underlined sequences represent the constant primer regions oneach sequence. FIG. 2B: Aptamer sequences were truncated on the basis ofsecondary structure predictions. The secondary structure prediction onthe left is of the M49 aptamer (SEQ ID NO: 1), and the secondarystructure prediction on the right is of the M52 aptamer (SEQ ID NO: 2).The M49-T1 (SEQ ID NQ-L 2 M52-T1 (SEQ ID NO: 13), and M52-T2 (SEQ ID NO:14) aptamer sequence truncations are indicated by the respective barscrossing SEQ ID NO: 1 (for M49-T1) and SEQ ID NO: 2 (for M52-T1 andM52-T2) s shown. FIG. 2C: Truncated aptamers were added to 5 dayallogenic-MLC with an equal number of responder C57BL/6 splenocytes andirradiated BALC/c stimulator cells. CTLs were assayed by monitoring ⁵¹Crrelease from P815 mastocytoma target cells incubated for 5 hours in a25:1 effector to target culture. Truncated sequences M52-T1 and M49-T1suppress CTL induction with efficacy equivalent to full length aptamers.Samples were tested in triplicate and shown is mean±SEM (*P<0.01, NS=notsignificant). Graph is a representative from 3 independent experiments.

FIGS. 3A, 3B, and 3C show Modification and Purification of DNA aptamerswith a 20 kDa PEG. FIG. 3A: DNA aptamers were synthesized with a 5′amine with a C6 spacer and reacted with an excess of monofunctionalNHS-PEG overnight. FIG. 3B: FPLC size exclusion chromatography using aSuperdex75 column was used to purify the crude reaction (top) to yieldpurified full length PEGylated aptamers (bottom). Absorbance (UV260 nm)was used to track elution of free nucleic acid and nucleic acidconjugates. The first peak at 7.93 mL corresponds to PEG-Aptamer, and9.66 mL peak corresponds to unreacted aptamer. FIG. 3C: Free aptamer(1), crude PEG-aptamer reaction mix (2), and FPLC purified PEG-aptamer(3) were run on an 8% polyacrylamide gel and visualised by silver stainto detect nucleic acids.

FIGS. 4A, 4B, and 4C show PEG conjugated M49 and M52 function act asCD200R1 agonists. FIG. 4A: PEGylated and non-PEGylated aptamers (7.5g/mL nucleic acid) were added to 5 day allogeneic-MLC of C57BL/6responder cells with an equal number of irradiated BALB/c stimulatorcells and CTL induction monitored by measuring P815 mastocytoma lysis.PEG-M49 and PEG-M52 suppress CTL induction by ˜60%. Each bar representsmean±SEM. *P<0.01 relative to no treatment, NS=not significant. FIG. 4B:To monitor aptamer induced phosphorylation of the CD200R1 cytoplasmictail, HEK293 cells transfected to express CD200R1 were incubated for 30minutes with PEG-M49 or PEG-M52. FIG. 4C: Cells were lysed and CD200R1immunoprecipitated and run on a western blot developed with an antibodyspecific to phosphorylated-CD200R1 cytoplasmic tail. Supernatant from aCD200 expressing cell was used as a positive control. (+) indicatesaddition of 5 uM PEG-M49, PEG-M52, or CD200+ supernatant, whereas (−)indicates media only. Shown is a representative of two independentexperiments.

FIGS. 5A and 5B show PEG-M49 and PEG-M52 function in-vivo. FIG. 5A:Experimental outline of in-vivo experiments. C57BL/6 mice were treatedover 12 days with intravenous tail vein injections of 151 μg PEG-M49,PEG-M52, PEG-cApt, or CD200Fc (positive control) in 72 intervals withlow-dose 0.5 mg/kg rapamycin. FIG. 5B: After 14 days mice weresacrificed and splenocytes used as responder cells in 5 day allogeneicMLC assays during which the treatment molecule was (white) or was not(grey) added in culture. In-vivo administration of PEG-M49 and PEG-M52in combination with rapamycin suppressed CTL induction without furtheraddition of aptamer in culture (P<0.01). Shown is a representative oftwo independent experiments.

FIGS. 6A and 6B show PEG-M49 and PEG-M52 prolong survival oftransplanted murine skin grafts. FIG. 6A: Experimental outline ofexperiment. C57BL/6 mice (n=6) received BALB/c skin allografts on day 0and were treated in 72 hr intervals with PBS (control) or 15 μgPEG-cApt, PEG-M49. PEG-M52 or CD200Fc (positive controls) in combinationwith low dose (0.5 mg/kg) rapamycin. Injections were given intravenouslyand graft survival monitored by a blinded investigator. FIG. 6B:Treatment with PEG-M49 and PEG-M52 significantly extended graft survival(P<0.05, Mantel-Cox Test) relative to control. Arrow represents the timeof last treatment (day 15). Shown is a representative of two independentexperiments.

FIGS. 7A and 7B show PEG-M49 and PEG-M52 but not PEG-cApt stimulatephosphorylation of the C-terminal tail of CD200R1. HEK293 expressingCD200R1 cells were incubated with indicated concentrations of PEG-M49,PEG-M52, PEG-cApt, or CD200 positive cell supernatant for 30 minutes.CD200R1 was immunoprecipitated and detected on western blot with anantibody specific to phosphorylated CD200R1 C-terminal tail.Phosphorylation was detected at all concentrations of PEG-M49 or M52(FIG. 7A and FIG. 7B) but not with PEG-cApt (FIG. 7B). (+) indicatesaddition of CD200+ supernatant, (−) indicates media alone. Shown is arepresentative from two independent experiments.

FIGS. 8A, 8B, and 8C show Cross Species Anti-CD200R Aptamers Identifiedby SELEX. FIG. 8A: At cycle 15 enriched SELEX pools towards both humanand murine CD200R recombinant proteins were sequenced using IonTorrentNext-Generation Sequencing. Thirteen sequences were identified to beenriched in both pools and labelled CCS1-13 accordingly (CD200R1 crossspecies). FIGS. 8B and 8C: Each aptamer was tested for suppression ofCTL induction in both human (FIG. 8B) and mouse (FIG. 8C) 5 dayallogenic mixed leukocyte cultures (MLC). Aptamers were added to MLC at10 uM. CTLs were identified by monitoring ⁵¹Cr release from loadedtarget cells at a 30:1 effector to target ratio. CCS.13 showed at least50% suppression of CTL induction in both human and mouse allogeniccultures and was chosen for further evaluation.

FIG. 9 shows conjugation of 20 kDa PEG to the 5′ terminus of CCS.13 doesnot inhibit functional activity. Monofunctional Polyethylene glycol(PEG) was conjugated to a 5′ amine modified aptamer with a C6 spacerovernight. Full length conjugated aptamers were purified using SEC-FPLCwith a Superdex-75 column. PEG-CCS.13 along with a scrambled controlsequence (PEG-cApt) was added at 3 ug/mL to 5 day human and mouseallogenic MLC to monitor suppression of CTL induction. CTLs were assayedby monitoring ⁵¹Cr release from loaded target cells at a 30:1 effectorto target ratio in 5 hours. PEG-CCS13, but not PEG-cApt significantly(*P<0.05) suppressed CTL induction in human and mouse allogenic MLC.

FIGS. 10A and 10B show PEG-CCS.13 functions in-vivo. FIG. 10A:Experimental outline of in-vivo experiment. C57BL/6 mice were treatedover 12 days with intravenous tail vein injections of 15 μg PEG-CCS.13.PEG-cApt, or CD200Fc (positive control) and low dose (0.5 mg/kg) in 72intervals. After 14 days mice were sacrificed and splenocytes used asresponder cells in 5 day allogeneic MLC assays. As shown in FIG. 10B,in-vivo administration of PEG-CCS.13 in combination with rapamycinsuppressed CTL induction without further addition of aptamer in culture(*P<0.01), whereas rapamycin alone or with PEG-cApt did not (NS=notsignificant).

FIGS. 11A and 11B show PEG-CCS.13 but not PEG-cApt prolong survival oftransplanted murine skin grafts. FIG. 11A: Experimental outline ofexperiment. C57BL/6 mice (n==6) received BALB/c skin allografts on day 0and were treated in 72 hr intervals with PBS (control) or 15 μgPEG-cApt, PEG-M49, PEG-M52 or CD200Fc in combination with low dose (0.5mg/kg) rapamycin. Injections were given intravenously and graft survivalmonitored by a blinded investigator. FIG. 11B: Treatment with PEG-CCS.13in combination with rapamycin significantly extended graft survival(P<0.05, Mantel-Cox Test) relative to PBS, or PEG-cApt with rapamycin.Arrow represents the time of last treatment (day 15).

FIG. 12 shows CCS acts as a CD200R1 agonist by inducing phosphorylationof the CD200R1 cytoplasmic tail. To monitor aptamer inducedphosphorylation of the CD200R1 cytoplasmic tail HEK293 cells transfectedto stably express mouse CD200R1 were incubated with a positive controlCD200R1 agonistic aptamer M49 (1.5 μM), a negative control aptamer cApt(3 μM), or full length (FL) or truncated versions (T1, T2) of theCD200R1 cross species aptamer CCS13 (all 3 μM). After a thirty minuteincubate the cells were subsequently lysed and phosphorylation of theCD200R tail detected by western blot using an antibody specific to mousephosphorylated-CD200R1 cytoplasmic tail. Treatment with CD200Fc (3.3 μM)or media alone were used as additional positive and negative controlsrespectively. Notably, full-length CCS13 but not its truncated versionswere capable of inducing phosphorylation of the CD200R1.

DETAILED DESCRIPTION

Disclosed are a series of aptamers which bind to murine CD200R1 andstimulate immune inhibitory signalling. These agonistic aptamers(termed, herein, M49, M52, and CCS13) exhibit in vitro immunosuppressiveproperties as measured by suppression of cytotoxic T-lymphocyte (CTL)induction in allogenic-mixed lymphocyte cultures (allo-MLC).Importantly, PEGylated conjugates of these aptamers retain bioactivityin-vitro and function as potent immunosuppressants when administeredin-vivo. The therapeutic potential of agonistic CD200R1 aptamers isdemonstrated—intravenous administration of PEG-M49 and PEG-M52 prolongsthe survival of murine skin allografts to a similar extent as CD200.Fc.CCS13 was also shown to be a cross-species aptamer, having function inboth humans and mice. The aptamers showed to suppress CTL induction in 5day mixed leukocyte culture (MLC) and cause rapid phosphorylation of theCD200R1 cytoplasmic tail thereby initiating immune inhibitorysignalling. PEGylated forms of these aptamers were synthesized and showsignificant in-vivo immunosuppression. In a murine model oftransplantation, intravenous injection of PEGylated aptamers enhancedsurvival of allogeneic skin grafts as effectively as a soluble CD200Fc.As DNA aptamers exhibit inherent advantages over conventionalprotein-based therapeutics including low immunogenicity, ease ofsynthesis, low cost, and long shelf life, the disclosed CD200R1 specificDNA aptamers are a useful and safe non-steroidal anti-inflammatorytherapeutic agent.

CD200R11 aptamers are disclosed at Table 1 (M49, M52, CCS8 and CCS13,having as their nucleotide sequences SEQ ID NOs 1, 2, 16 and 3,respectively); all show high specificity and binding affinity toCD200R1, and in two cases (CCS8 and CCS13) has been shown capable ofcross-species agonistic activity. The CD200R1-specific regions of theseaptamers are shown herein at Table 2, (TM49, TM52, TCCS8 and TCCS13),having as their nucleotide sequences the SEQ ID NOs.: 4, 5, 15 and 6,respectively.

TABLE 1 CD200R1 specific DNA aptamers M49:GACGATAGCGGTGACGGCACAGACGGACGTGACATGCTTGACCAACTCGCCGTATGCCGCTTCCGTCCGTCGCTC (SEQ ID NO.: 1) M52:GACGATAGCGGTGACGGCACAGACGTTTATTACCATTATGCCTATGTAACGTATGCCGCTTCCGTCCGTCGCTC (SEQ ID NO.: 2) CCS8:GACGATAGCGGTGACGGCACAGACGCCCCTCCGAGTGATATGTAATCCTACGTATGCCGCTTCCGTCCGTCGCTC (SEQ ID NO: 16) CCS13:GACGATAGCGGTGACGGCACAGACGCACCGCTCTTATGCCACCATTTTCACGTATGCCGCTTCCGTCCGTCGCTC (SEQ ID NO.: 3)

TABLE 2 CD200R1 - specific regions of the CD200R1 aptamers TM49:GACGTGACATGCTTGACCAACTCGC (SEQ ID NO.: 4) TM52:TTTATTACCATTATGCCTATGTAA (SEQ ID NO.: 5) TCCS8:CCCCTCCGAGTGATATGTAATCCTA  (SEQ ID NO.: 15) TCCS13:CACCGCTCTTATGCCACCATTTTCA (SEQ ID NO.: 6)

Example 1: Aptamer Selection

ssDNA aptamers specific towards murine CD200R1 were identified using thePCR-based Systematic Evolution of Ligands by Exponential Enrichment(SELEX) method (Ellington and Szostak, Nature, 1990 Aug.30:346(6286):818-22; Tuerk and Gold, Science 1990 Aug. 3249(4968):505-10; Bock et al., Nature 1992 Feb. 6:355(6360):564-6, allincorporated herein by reference). A 25 nucleotide long random syntheticoligonucleotide library flanked by 25-base long 5′ and 3′ primer regions(5′-G ACGATAGCGGTGACGGCACAGACGNNNNNNNNNNNNNNNNNNNNNNNNNCGTATGCCGCTTCCGTCCGTCGCTC-3′, SEQ ID NO.: 7) was synthesized byIntegrated DNA Technologies (IDT) along with the corresponding primersequences (Forward 5′-GACGATAGCGGTGACGGCACAGACG-3′ (SEQ ID NO.: 8) andReverse 5′ GAGCGACGGACGGAAGCGGCATACG-3′ (SEQ ID NO.: 9)). A 4 nmolaliquot of the library representing ˜2.5×10¹⁵ sequences was adsorbedonto MagneHis Ni-Particles (Promega) at 37° C. for 1 hr to removesequences which bound to the solid support. The resulting sub-librarywas incubated for 1 hr at 37° C. with 10 μg of a recombinant HIS-taggedmurine CD200R1 protein immobilized on MagneHis Ni-Particles suspended in1 mL phosphate buffered saline (PBS, pH 7.4). Unbound and weakly boundsequences were removed by washing the beads with PBS for 5 minutes andprotein-aptamer complexes were eluted with PBS containing 0.5Mimidazole. Aptamers were recovered using the Qiagen Nucleotide Removalkit following manufacturer's recommendations and the ssDNA pool wasamplified for the next round of selection using asymmetric PCR at a 10:1forward:reverse primer ratio. Fifteen rounds of selection were performedwith the selection stringency increasing as the concentration of CD200R1was halved every three rounds while simultaneously increasing the numberof wash steps. After the 15^(th) cycle, selected DNA aptamers werecloned into pCR4-TOPO vector (Life Technologies) and sequenced.

Over 20 DNA aptamer sequences specifically recognizing a murine CD200R1recombinant protein were identified after 15 rounds of SELEX screens.These 75-base long sequences along with a scrambled control aptamer(cApt) (GACGATAGCGGTGACGGCACAGACGTCCCGCATCCTCCGCCGTCCCGACCCGTATGCCGCTTCCGTCCGTCGCTC (SEQ ID NO.: 10), containing specific regionTCCCGCATCCTCCGCCGTGCCGACC (SEQ ID NO.: 11)) were synthesized andsystematically screened for CD200R1 agonistic activity by evaluatingtheir capability to suppress the induction of cytotoxic T-lymphocyte(CTL) in 5 day allo-mixed lymphocyte cultures. Aptamer-inducedsuppression of CTL induction was monitored using a chromium releaseassay of labeled P815 mastocytoma serving as target cells for CTL lysis.Four aptamers M21, M48, M49, and M52 (FIG. 1A) displayed CD200R1agonistic properties (FIG. 1B). Specifically aptamers M49 and M52suppressed CTL induction at levels comparable to the soluble CD200Fcligand with less than 5% CTL specific lysis of P815 cells occurring ataptamer concentrations ≥7.5 μg/mL. M49 and M52 were chosen for furtherevaluation.

The 75-base long M49 and M52 aptamer sequences were further truncatedbased on their predicted secondary structure derived from mfold software(FIGS. 2A and 2B). M49 retained agonistic activity when truncated to aminimal size of 55-bases TrM49 (SEQ ID NO.: 12) while the optimalactivity for M52 was retained down to a length of 44 bases TrM52 (SEQ IDNO.: 13) (FIG. 2C).

Example 2: Allogeneic Mouse Mixed Lymphocyte Culture (Allo-MLC)

Agonistic CD200R1 aptamers were identified and evaluated for theirability to suppress cytotoxic T-lymphocyte (CTL) induction in 5 dayallo-MLC. Briefly, 2.5×10⁵ C57BL/6 responder splenocytes were incubatedwith an equal number of irradiated BALB/c stimulator cells in thepresence of synthetic aptamers, PEGylated aptamers, or CD200Fc for 5days. CTL induction was assayed by monitoring the release of ⁵¹Cr fromloaded P815 mastocytoma target cells over a 5 hour time period at a 25:1effector-to-target ratio.

Example 3: PEGylation of DNA Aptamers

The 5′ termini of aptamer M49 (SEQ ID NO.: 4), M52 (SEQ ID NO.: 5), andthe control aptamer cApt (SEQ ID NO.: 10) were modified with a 20 kDapolyethylene glycol (PEG) moiety to increase their circulatoryhalf-life, as shown schematically in FIG. 3A. Briefly, a 5′ amino groupwith a hexylamine arm was incorporated into each DNA aptamer duringsynthesis (IDT). A 100-molar excess of mPEG-succinimidyl glutarate esterpowder (Creative PEGWorks, Winston Salem, N.C., USA) was added stepwiseover a period of 10 hours to 25 μM solutions of the modified aptamersdissolved in 100 mM NaHCO₃/CH₃CN (1:1 pH 8.5). The PEGylated aptamerswere purified by ultrafiltration using Amicon Ultra Centrifugal Filterswith a 30 kDa MWCO (Millipore) and size exclusion fast protein liquidchromatography (FPLC) using a Superdex 75 10/300 column (GE Healthcare)with 100 mM NH₄CO₃ as eluent. Purified PEGylated-aptamer conjugates werelyophilized and resuspended in sterile PBS for subsequent experiments.Purity of the final PEGylated products were confirmed by size exclusionFPLC (FIG. 3B) and polyacrylamide gel electrophoresis (FIG. 3C).

The PEGylated aptamers PEG-M49, PEG-M52, and PEG-cApt were compared tounconjugated aptamers for their capability to suppress CTL induction inallogeneic-MLC. Both PEG-M49 and PEG-M52 suppressed CTL induction to agreater extent than M49 and M52 (FIG. 4A), confirming that PEGylationdid not disrupt the structure and function of these aptamers.

Example 4: Detection of CD200R1 Phosphorylation

Intracellular phosphorylation of CD200R1 in response to PEG-M49,PEG-M52, and PEG-cApt was detected using a rabbit polyclonal antibodyspecific to the phosphorylated cytoplasmic tail of CD200R1. HEK-293cells stably expressing CD200R1 were serum starved in OptiMEM media(Life Technologies) for 3 hrs followed by incubation for 30 min with 2.5μM PEG-M49, PEG-M52, PEG-cApt, or a CD200 positive cell lysate (positivecontrol) in OptiMEM. Cells were washed in PBS, lysed in RIPA buffer withprotease inhibitor, and CD200R1 immunoprecipitated using an anti-CD200R1(clone 2A10) monoclonal antibody (overnight 4° C.) and Protein G agarosebeads (Pierce). The phosphorylated form of CD200R1 was detected bywestern blot using the rabbit polyclonal antibody (1:1000 dilution) andanti-rabbit HRP (1:15,000 dilution).

The immediate signalling event following CD200:CD200R1 ligation is thephosphorylation of the tyrosine residue in the NPXY motif on theC-terminal cytoplasmic tail of CD200R1. The phosphorylated NPXY motifinteracts with adaptor proteins thereby transducing immune inhibitorysignalling. To confirm that the suppression of CTL induction observed inour allo-MLC assays was indeed a consequence of aptamer-induced CD200R1signalling, we verified whether PEG-M49 and PEG-M52 could induce thephosphorylation of this motif. HEK-293 cells were stably transfected toexpress murine CD200R1 (FIG. 4B) and treated with aptamers PEG-M49 andPEG-M52. The phosphorylation of CD200R1 was detected using aphosphospecific antibody. Both PEG-M49 and PEG-M52 induced the rapidphosphorylation of the C-terminal tail of CD200R1 (FIG. 4C). There wasno detectable signal from medium alone or PEG-cApt (FIG. 7) confirmingthat the identified aptamers signal through CD200R1 in a similar mannerto CD200.

Example 5: Activity of PEGylated Aptamers In-Vivo

To stimulate an immune response C57BL/6 mice received BALB/c skinallografts (Day 0) followed by five tail vein injections of 15 μgPEG-M49, PEG-M52, PEG-cApt, or CD200Fc dissolved in 0.3 mL PBS, pH 7.4every 72 hours over 12 days in combination with low dose (0.5 mg/kg)rapamycin administered intraperitoneally every 48 hours (shownschematically in FIG. 5A). On day 14, mice were sacrificed andsplenocytes used as responder cells in 5 day ex-vivo allo-MLC with orwithout further aptamer addition in vitro.

CTL induction was significantly suppressed by treating animals withPEG-M49, PEG-M52, or CD200Fc alone but not with PEG-cApt (P<0.01)(Figure SB, grey bars). Exposure of circulating lymphocytes to PEG-M49and PEG-M52 both in-vivo and after their recovery (in-vitro) did notsignificantly improve the suppression of CTL induction as compared toin-vivo alone (FIG. 5B). This finding suggests that the administrationof PEGylated CD200R1 agonistic aptamers is sufficient to down regulateimmune responses in-vivo and that such aptamers may serve asanti-inflammatory agents for diseases in which CD200:CD200R1 signallingis implicated.

Example 6: Allogeneic Skin Graft Transplantation

PEG-M49 and PEG-M52 were evaluated for their capability to prolongsurvival of allogeneic murine skin grafts. C57BL/6 mice (n=6) receivedBALB/c skin allografts (Day 0) prior to receiving 6 tail vein injectionsof 15 μg PEG-M49, PEG-M52, PEG-cApt or CD200Fc in 72 hr intervals over15 days in combination with low dose (0.5 mg/kg) rapamycin administeredintraperitoneally every 48 hrs. Graft survival was monitored daily by ablinded investigator.

Rapamycin at this dosage has been shown to have no effect on graftsurvival when administered alone. Treatment with PEG-M49 and PEG-M52significantly extended allograft survival as compared to PBS or PEG-cAptgroups (FIG. 6B) (P<0.05, Mantel-Cox Test). Interestingly, at the timeof last injection (Day 15) only 16% of mice receiving PEG-M49 or PEG-M52had rejected the allografts. After this time point, differences betweenCD200Fc and PEGylated aptamers may be linked to differingpharmacokinetic profiles as the circulatory half-life of PEGylatedaptamers and therefore their bioavailability may differ from the proteinCD200Fc.

Example 7: Cross Species CD200R1 Agonistic Aptamers

The existing enriched libraries from SELEX to human and mouse CD200R1were deep sequenced using IonTorrent and compared to each other toidentify overlapping sequences. These sequences were synthesized andscreened for CD200R1 agonistic activity (suppression of CTL in allo-MLC)with cells of both human and mouse origin. The identified agoniststermed CCS13 and CCS8 (SEQ ID NOs.: 3 and 16, respectively, see Table 1,above) were PEGylated and activity verified by human and mouse allo-MLC.To evaluate in-vivo immunosuppression C57BL/6 mice received BALB/callografts (Day 0) and were treated with 15 ug PEGylated aptamers orCD200.Fc in combination (72 hrs, i.v) with low dose rapamycin (0.5mg/kg, 36 hrs, i.p.). Immune responses were monitored at Day 15 byex-vivo allo-MLC. Lastly therapeutic potential was evaluated bymonitoring the capability of PEG-CCS13 and PEG-CCS8 to prolong survivalof the C57BL/6 allografts using the same treatment regimen describedabove to a total of 15 days. Graft survival was monitored by a blindedinvestigator.

Aptamers CCS13 and CCS8 induced potent suppression of CTL induction(<50% specific lysis) in both human (FIG. 8B) and mouse allo-MLC (FIG.8C). CCS13 and CCS8 were PEGylated and found to retain activity in 5-dayin both human and mouse allo-MLC (FIG. 9). In-vivo administration ofPEG-CCS8 and PEG-CCS13 significantly suppressed immune activity asmeasured by ex-vivo allo-MLC. (FIG. 10). Finally, PEG-CCS8 and PEG-CCS13were found to significantly prolong allograft survival (FIG. 11).

Thus the aptamers CCS8 and CCS13 potently induced immunosuppression inboth human and mouse allo-MLC. Furthermore PEGylated CCS13 and CCS8retained this ability and functioned in-vivo to suppress immune responseand prolong allograft survival.

Example 8: Detection of CD200R1 Phosphorylation

Intracellular phosphorylation of CD200R1 in response to M49, as well asfull length and truncated versions of CCS13 was detected using a rabbitpolyclonal antibody specific to the phosphorylated cytoplasmic tail ofCD200R1. HEK-293 cells stably expressing murine CD200R1 wereserum-starved in OptiMEM (Life Technologies, Burlington, Canada) mediumfor 3 hrs and subsequently incubated for 30 min in OptiMEM mediumcontaining either M49 (1.5 μM), negative control aptamer cApt (3 μM),CD200Fc (3.5 μM), or full length (FL) or truncated (T1, T2) versions ofCCS13 (3 μM). Cells were washed with PBS and lysed in RIPA buffer (150mM NaCl, 1.0% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mMTris, pH 8.0) containing 50 mM NaF, 1 mM Na3VO4, and proteaseinhibitors. Phosphorylated and unphosphorylated forms of CD200R1 wererecovered by immunoprecipitation using an anti-CD200R1 (clone 2A10)monoclonal antibody (overnight 4° C.) and Protein G agarose beads(Pierce). The phosphorylated form of CD200R1 was detected by westernblot using the rabbit polyclonal antibody (1:1000 dilution) andanti-rabbit HRP (1:15.000 dilution) (FIG. 12). Full-length CCS13 but notits truncated versions were capable of inducing phosphorylation of theCD200R1.

Example 9: Treatment of Asthma with an Aerosolized, Nasally or OrallyInhaled Aptamer

Methods of administering small molecules via an intranasal route, or vianasal or oral inhalation, are known. For example, a saline-basedsolution comprising the active small molecule can be administered as amist; dry powder can be administered via a known dry powder inhaler.

In certain embodiments, administration of an effective amount of aCD200R1 agonist on its own, for example, an aptamer, such as a PEGylatedaptamer (e.g., PEG-CCS13), is effective in treating asthma. In aparticular embodiment, a solution of an effective amount of PEG-CCS13 isprepared using standard inhalation carriers, such as a saline mist,which can be administered intranasally or via nasal or oral inhalationfor the treatment of asthma. Alternatively, a dry powder form ofPEG-CCS13 is administered by a known dry powder inhaler.

In an alternative embodiment, PEG-CCS13 is added to an already known andexisting treatment for asthma, such as a corticosteroid. It is expectedthat such a combination therapy may be particularly effective intreating asthma, for example, by having improved efficacy or asynergistic effect over administration of a corticosteroid alone, sincea corticosteroid has a different and distinct mechanism of action thanPEG-CCS13.

In another embodiment, a single dose of PEG-CCS13, providedintranasally, produces significant alleviation of asthma symptoms in anindividual with asthma.

In yet another embodiment, a CD200R1 agonist, for example CCS13,conjugated to one or more active ingredients of known asthma medicines,provides an increase in the effectiveness of such medicines, bytargeting those medicines to an appropriate location for improvedeffect.

The invention claimed is:
 1. A method of treating asthma, comprisingadministering to a subject in need thereof an effective amount of anaptamer or oligonucleotide that binds to a CD200R1 target, wherein theaptamer or oligonucleotide is selected from the group consisting ofCCS13 (SEQ ID NO:3), CCS8 (SEQ ID NO:16), TCCS8 (SEQ ID NO: 15), andTCCS13 (SEQ ID NO:6).
 2. The method of claim 1, wherein the aptamer oroligonucleotide comprises at least one chemical modification.
 3. Themethod of claim 2, wherein the modification is incorporation of amodified nucleotide.
 4. The method of claim 2, wherein the modificationis pegylation.
 5. The method of claim 4, wherein the pegylation is alinear polymer of polyethylene glycol.
 6. The method of claim 2, whereinthe modification is 3′ and 5′ capping.
 7. The method of claim 2, whereinthe modification is conjugation to a high molecular weight,non-immunogenic compound; conjugation to a lipophilic compound; orconjugation to another oligonucleotide.
 8. The method of claim 7,wherein the another oligonucleotide is an siRNA, an antisenseoligonucleotide, a ribozyme, or a DNA/RNA chimera.
 9. The method ofclaim 1, wherein the aptamer or oligonucleotide is conjugated orotherwise associated with a cytotoxic agent.
 10. The method of claim 1,wherein the aptamer or oligonucleotide is an aptamer- oroligonucleotide-drug conjugate comprising the aptamer or oligonucleotideand a cytotoxic drug.
 11. The method of claim 1, wherein the aptamer oroligonucleotide is an aptamer- or oligonucleotide-protein conjugatecomprising the aptamer or oligonucleotide and a therapeutic protein. 12.The method of claim 1, wherein the aptamer or oligonucleotide is anaptamer- or oligonucleotide-radionuclide conjugate comprising theaptamer or oligonucleotide and a radionuclide.
 13. The method of claim1, wherein the aptamer or oligonucleotide is an aptamer- oroligonucleotide-metal chelator conjugate comprising the aptamer oroligonucleotide and a metal chelator.
 14. The method of claim 1, whereinthe aptamer or oligonucleotide is an aptamer- oroligonucleotide-nanostructure conjugate comprising the aptamer oroligonucleotide and a nanostructure.
 15. The method of claim 1, whereinthe aptamer or oligonucleotide is an aptamer- oroligonucleotide-chromophore conjugate comprising the aptamer oroligonucleotide and a chromophore.
 16. The method of claim 1, whereinthe aptamer or oligonucleotide is a pegylated CCS13 (SEQ ID NO: 3). 17.A pharmaceutical composition for the treatment of asthma comprising anaptamer or oligonucleotide selected from the group consisting of CCS13(SEQ ID NO:3), CCS8 (SEQ ID NO:16), TCCS8 (SEQ ID NO: 15), and TCCS13(SEQ ID NO:6) and a therapeutically acceptable carrier.
 18. Thepharmaceutical composition of claim 17 wherein the aptamer oroligonucleotide is pegylated.
 19. The pharmaceutical composition ofclaim 18 wherein the aptamer or oligonucleotide is a pegylated CCS13(SEQ ID NO: 3).